人间皮素基因中Wnt/β-catenin通路调控元件分析及在卵巢癌细胞中启动子区鉴定

Bioinformatics analysis of Wnt/β-catenin pathway related regulatory elements in human mesothelin gene and identification of promoter in ovarian cancer cells

目的分析人间皮素基因启动子区中Wnt/β-catenin信号通路相关转录因子T细胞因子/淋巴细胞增强因子(T cell factor/lymphoid enhancer factor,TCF/LEF)结合位点,鉴定间皮素基因在卵巢癌细胞表达相关核心启动子区。方法采用生物信息学方法分析人间皮素基因启动子区域内可能的TCF/LEF转录因子结合位点。克隆人间皮素基因5'端附近1 764 bp启动子序列,对该片段做5'端的不同截短,克隆至p GL3-basic报告基因载体,分别转染人卵巢癌细胞系SKOV3和3-AO,双荧光酶报告基因系统检测不同启动子片段活性。结果人间皮素基因启动子区含有多个潜在的TCF/LEF结合位点。成功克隆扩增间皮素基因启动子区-1456～+308、-164～+308、+47～+308三个片段,经测序证明无误,构建p GL3载体。转染SKOV-3与3-AO细胞后,双荧光酶报告基因系统检测显示-1456～+308与-164～+308片段在两个细胞系中均有较高启动活性,+47～+308片段活性较前两者显著下降(P均<0. 01)。结论含有TCF/LEF转录因子结合位点的-164～+47序列是卵巢癌中间皮素基因表达的核心启动子区,为进一步观察卵巢癌中间皮素基因的表达调控机制奠定基础。

Purpose To analyze the Wnt/β-catenin signaling pathway related TCF/LEF binding sites in human mesothelin gene and to identify the core promoter region of the gene in the ovarian cancer cells.Methods The possible TCF/LEF transcription factor binding sites in the promoter region of human meosothelin gene were analyzed by bioinformatics method.The 1 764 bp promoter sequence near the 5'end of the human mesothelin gene were cloned.The fragments was truncated differently at the 5'end and cloned into pGL3-basic report gene vector and transfected into human ovarian cancer cell lines SKOV-3 and 3-AO.The activity of diffetent promoter fragmentis was detected by double luciferase reporter gene system.Results There were multiple potential TCF/LEF binding sites in the promoter region of the human mesothelin gene.Three fragments of mesothelin gene promoter region-1456-+308、-164-+308、+47-+308 were cloned and amplified successfully,the pGL3 vector was constructed by sequencing.After transfection of SKOV-3 and 3-AO cells,the double luciferase reporter gene system showed that the-1456-+308 fragments and-164-+308 framents had high promoting activity in both cell lines,and the activity of+47-+308 fragments was significantly lower than that of the former two cell lines(P<0.01).Conclusion The-164-+47 sequence containing TCF/LEF transcription factor binding site is the core promoter region of mesothelin gene in ovarian cancer,which lays a foundation for further study on the regulation mechanism of mesothelin gene expression in ovarian cancer.